The ENTH R114A mutant

There are different regions within the ENTH domain involved in protein-protein and protein-lipid interactions. Especially the amino acid R114 was found to interact with the membrane and to be involved in the formation of ENTH homo-oligomers.^26,110 Lai et al. performed coarse-grained molecular dynamics (CG-MD) simulation and EPR ex-periments with the ENTH domain. Their results indicated an interaction of the amino acid R114, located in the unstructured loop between helix 6 and helix 7, with the membrane.¹¹⁰ This interaction further recruits ENTH and stabilizes the ENTH mem-brane docking geometry. Hence, the amino acid R114 can be involved in the cluster formation of ENTH.

In order to investigate the role of the amino acid R114 in the formation of clusters, as observed in the presence of PS (Figure 4.17), RIfS and AFM experiments were per-formed with the mutant R114A. The results showed a decreased dissociation coeffi-cient of the mutant on POPC/POPS/PIP2 bilayers and a higher protein occupancy on the membrane compared to bilayers lacking PS. Furthermore similar surface occu-pancies of ENTH R114A and ENTH wt were determined indicating that the mutant is still binding to PIP2 doped membranes. However, the dissociation constant of the mu-tant significantly increased compared to the wt showing the contribution of the amino acid R114 in the binding process of ENTH. The obtained values for the ENTH wt and mutant are listed in Table 5.1.

5 Discussion

114

Table 5.1: Summary of the fit results of the Langmuir adsorption isotherms for ENTH wt and ENTH R114A. The values are given as parameter ± SE.

lipid

In general, the dissociation constants lie within the same range as the values found in literature (cf. Appendix, Table 10.3). Stahelin et al. mutated the ENTH domain at dif-ferent positions. Mutations, like L6Q and M10Q strongly affected the binding affinity and the penetration ability into monolayers as these amino acids were localized in the α0 helix.²⁵ The R114A mutant in turn exhibited a decreased dissociation constant compared to the wt, but showed a similar penetration ability into POPC/POPE/PIP2

(77:20:3) as the ENTH wt. This amino acid is not located at the PIP2 binding site, but the close contact to the membrane facilitates the contribution in non-specific electro-static interactions.^23,25,110

Lack of the positively charged protein residue (R114) or the negatively charged PS in the membrane led to a decreased binding affinity (Figure 4.21) and hence support the assumption that the amino acid R114 is involved in further recruitment of ENTH when PS was present. As the occupancy of ENTH R114A in presence of PS was similar to that of ENTH wt (cf. Table 5.1), it seems likely that still interactions of PS and the mutant occur in a PIP2 dependent manner, but ENTH clustering is prevented (Figure 4.22). Although the KD of the mutant is higher as the value for the ENTH wt, the mo-bility of single ENTH R114A molecules seemed to be higher than the momo-bility of the ENTH wt clusters, hence the resolution of individual ENTH R114A monomers (and also individual ENTH wt monomers in the absence of PS, Figure 4.17) in contact mode was not possible.

Previous studies revealed the contribution of the amino acid R114 of ENTH in recruit-ment and membrane deformation. Kroppen showed that the mutation of this amino

5 Discussion

115 acid did not change the molecular structure of ENTH and still was binding to the mem-brane in presence of PIP2 and PS.²⁶ However, the ability to form tubular structures got lost proving the importance of this amino acid.²⁶ Moreover, co-sedimentation as-says revealed the contribution of R114 in homo-oligomer formation. To investigate if the mutant also prevents cluster formation on solid-supported membranes, AFM ex-periments were performed. In all cases (DOPC/DOPE/PIP2/TxR (64.9:30:5:0.1) and DOPC/DOPE/DOPS/PIP2/TxR (44.9:30:20:5:0.1)) homogenous and planar bilayers with a roughness of 152±7 pm (without PS) and 197±47 pm (with PS) were observed after the incubation with ENTH R114A. Even in the presence of PS no clusters ap-peared (Figure 4.22) meaning that the lack of the arginine in position 114 prevented the formation of clusters. This also shows that the presence of PS and the amino acid R114 are required for the cluster formation which also can indicate an interaction of PS and R114.

Yoon et al. already revealed that interactions between two ENTH monomers can oc-cur in the presence of PIP2 and PS.²⁸ Although they did not investigate the impact of PS, they performed vesicle binding studies with GUVs composed of POPC/POPE/POPS/PIP2/Rh-PE (46.5:30:20:0.5, Rh-PE = 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)) showing the appear-ance of tubulation induced by ENTH crowding. This was attributed to the interaction of K23 and E42 of two ENTH monomers. When the amino acids were mutated (K23E and E42K) the ENTH lost the ability to induce tubulations. Their vesicle binding stud-ies were supported by EPR studstud-ies which indicated an interaction of two α0 helices (ENTH wt) in an antiparallel manner meaning a well-ordered clustering of the pro-teins. In combination with the results observed in this thesis, a contribution of PS and R114 in the accessibility of the amino acids K23 and E42 for protein-protein interac-tions (Figure 5.9) seems likely.

5 Discussion

116

Figure 5.8: Orientation and interactions of two ENTH monomers in the (A) absence and (B) presence of PS. When PS is lacking less ENTH-membrane interactions occur. Due to the presence of PS interac-tions of ENTH with the membrane increase. Changed penetration depth and angles can lead to a facil-itated interaction of K23 and E42 resulting in cluster formation.

Yoon et al. furthermore determined the insertion depth of the ENTH helix (usually 13 Ų from the phosphate group) as well as the penetration angle of the helix (11° to the membrane normal) in the presence of PS.²⁸ Lack of PS and/or R114 may change the orientation of the helix in the membrane which could lead to a different accessi-bility of the amino acids K23 and E42 for protein-protein interactions. Consequently less or no clusters were formed preventing imaging by AFM due to the mobile char-acter of ENTH monomers (cf. chapter 5.1.2).

In summary, it can be stated that interactions of the amino acid R114 with the mem-brane or/and other ENTH monomers in the presence of PS are involved in the ENTH cluster formation.

5.2 Lipid dependent penetration ability of ENTH to lipid